The conserved B3 domain of VIVIPAROUS1 has a cooperative DNA binding activity.

The biochemical activities that underlie the genetically defined activator and repressor functions of the VIVIPAROUS1 (VP1) protein have resisted in vitro analysis. Here, we show that a glutathione S-transferase (GST) fusion protein, including only the highly conserved B3 domain of VP1, has a highly cooperative, sequence-specific DNA binding activity. GST fusion proteins that include larger regions of the VP1 protein have very low activity, indicating that removal of the flanking protein sequences is necessary to elicit DNA binding in vitro. DNA competition and DNase I footprinting analyses show that B3 binds specifically to the Sph element involved in VP1 activation of the C1 gene, whereas binding to the G-box-type VP1-responsive element is of low affinity and is nonspecific. Footprint analysis of the C1 promoter revealed that sequences flanking the core TCCATGCAT motif of Sph also contribute to the recognition of the Sph element in its native context. The salient features of the in vitro GST-B3 DNA interaction are in good agreement with the protein and DNA sequence requirements defined by the functional analyses of VP1 and VP1-responsive elements in maize cells.     

Yeast DNA polymerase--DNA primase complex; cloning of PRI 1, a single essential gene related to DNA primase activity.

The immunopurified yeast DNA polymerase--DNA primase complex is constituted by DNA polymerase I polypeptides and by three other protein species, called p74, p58 and p48, which we show to be immunologically unrelated. The gene encoding the p48 polypeptide has been identified by immunological screening of a lambda gt11 yeast genomic DNA library. Antiserum specific for p48 inhibits DNA primase, and immunoreactive, inhibitory antibodies are affinity-purified by the clone-encoded protein, thus relating the p48 polypeptide to DNA primase activity. The entire gene has been cloned, and the 1.45-kb p48 mRNA is overproduced in cells containing the gene in high copy number. Gene disruption and Southern hybridization experiments demonstrate that the p48 protein is encoded by a single gene and it performs an essential function.     

Yeast Rmi1/Nce4 controls genome stability as a subunit of the Sgs1-Top3 complex

Genome stability requires a set of RecQ-Top3 DNA helicase-topoisomerase complexes whose sole budding yeast homolog is encoded by SGS1-TOP3. RMI1/NCE4 was identified as a potential intermediate in the SGS1-TOP3 pathway, based on the observation that strains lacking any one of these genes require MUS81 and MMS4 for viability. This idea was tested by confirming that sgs1 and rmi1 mutants display the same spectrum of synthetic lethal interactions, including the requirements for SLX1, SLX4, SLX5, and SLX8, and by demonstrating that rmi1 mus81 synthetic lethality is dependent on homologous recombination. On their own, mutations in RMI1 result in phenotypes that mimic those of sgs1 or top3 strains including slow growth, hyperrecombination, DNA damage sensitivity, and reduced sporulation. And like top3 strains, most rmi1 phenotypes are suppressed by mutations in SGS1. We show that Rmi1 forms a heteromeric complex with Sgs1-Top3 in yeast and that these proteins interact directly in a recombinant system. The Rmi1-Top3 complex is stable in the absence of the Sgs1 helicase, but the loss of either Rmi1 or Top3 in yeast compromises its partner's interaction with Sgs1. Biochemical studies demonstrate that recombinant Rmi1 is a structure-specific DNA binding protein with a preference for cruciform structures. We propose that the DNA binding specificity of Rmi1 plays a role in targeting Sgs1-Top3 to appropriate substrates.     

Inhibition of APCCdh1 activity by Cdh1/Acm1/Bmh1 ternary complex formation

The anaphase-promoting complex (APC) is an essential E3 ubiquitin ligase responsible for catalyzing proteolysis of key regulatory proteins in the cell cycle. Cdh1 is a co-activator of the APC aiding in the onset and maintenance of G(1) phase, whereas phosphorylation of Cdh1 at the end of G(1) phase by cyclin-dependent kinases assists in the inactivation of APC(Cdh1). Here, we suggest additional components are involved in the inactivation of APC(Cdh1) independent of Cdh1 phosphorylation. We have identified proteins known as Acm1 and Bmh1, which bind and form a ternary complex with Cdh1. The presence of phosphorylated Acm1 is critical for the ternary complex formation, and Acm1 is predominantly expressed in S phase when APC(Cdh1) is inactive. The assembly of the ternary complex inhibits ubiquitination of Clb2 in vitro by blocking the interaction of Cdh1 with Clb2. In vivo, lethality caused by overexpression of constitutively active Cdh1 is rescued by overexpression of Acm1. Partially phosphorylated Cdh1 in the absence of ACM1 still binds to and activates the APC. However, the addition of Acm1 decreases Clb2 ubiquitination when using either phosphorylated or nonphosphorylated Cdh1. Taken together, our results suggest an additional inactivation mechanism exists for APC(Cdh1) that is independent of Cdh1 phosphorylation.     

Ribosomal DNA of the fly Sciara coprophila has a very small and homogeneous repeat unit

Summary In this report we show by hybridization of restriction fragments and by Miller spreads that the unit repeat of the fly Sciara coprophila is only 8.4 kb which is the smallest known for a multicellular eukaryote. The 8.4 kb EcoR1 fragment containing a complete unit of Sciara rDNA was cloned in pBR322, and mapped by the method of Parker (1977) and also by double digestion. The coding regions for 28S, 18S, and 5.8S RNA were localized by the method of Berk and Sharp (1977). From these data we conclude that the nontranscribed spacer, external transcribed spacer, and internal transcribed spacer are all shorter than in other organisms, thereby giving rise to the shorter overall rDNA repeat unit of Sciara.At least 90% of the Sciara rDNA repeats are homogeneous, with a length of 8.4 kb, but a 700 bp ladder of minor bands can also be found in digestions of total genome DNA. This profile of major and minor bands is identical between the X and X chromosomes, as seen by a comparison of several genotypes.There are only 45 rRNA genes per X chromosome of Sciara (Gerbi and Crouse, 1976). These can easily be counted by low magnification Miller speads which show that virtually all gene copies are actively being transcribed in the stage of spermatogenesis examined. This is the first demonstration for any reiterated gene family where all copies are shown to be simultaneously active.Present address same as last author     

Iron(III) binding in DNA solutions: complex formation and catalytic activity in the oxidation of hydrazine derivatives.

A strong interaction between iron(III) and calf thymus DNA at pH 7.4 was demonstrated in the present study by separation of the complex by column chromatography and by the slow kinetics of iron(III) removal from DNA by disodium-1,2-dihydroxybenzene-3,5-disulfonate (Tiron). An equilibrium constant of 2.1 x 10(14) was calculated by measurements of bound iron(III) by flame atomic absorption spectroscopy and assuming a one iron to two nucleotide stoichiometry. Graphic analysis of the interaction however, indicated that DNA has two binding sites for iron(III) characterized by a stoichiometry of one iron to 12 nucleotides and one iron to 2 nucleotides, and association constants of 4.8 x 10(12) and 2.3 x 10(11), respectively. The DNA-iron(III) complex isolated by column chromatography was shown to catalyze the oxidation of both 2-phenylethylhydrazine and methylhydrazine by spin-trapping experiments with alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN). By contrast, oxidation of 1,2-dimethylhydrazine was not catalyzed. Catalysis of 2-phenylethylhydrazine oxidation was confirmed by oxygen consumption studies. The results suggest that iron chelated to DNA may be significant in DNA damage induced by oxidizable chemicals.     

Nature of double-stranded DNA binding activity in seropositive rheumatoid arthritis: formation of low avidity DNA/rheumatoid factor/IgG/low density lipoprotein complexes

Sera from majority of patients with seropositive rheumatoid arthritis, which generally lacked detectable anti-double stranded DNA in Farr, Crithidia luciliae, and microcomplement fixation assays, exhibited high levels of dsDNA binding in the presence of 3.5% polyethylene glycol when using intrinsically labeled 3H-PM2 DNA as antigen. Except for SLE, such increased dsDNA binding was absent in normal and a variety of other disease sera, including those from patients with seronegative rheumatoid arthritis. In contrast to the situation in SLE, in which dsDNA binding is mediated by specific anti-DNA antibody, the increased dsDNA binding activity in seropositive rheumatoid arthritis was shown to be dependent upon complex low avidity interactions involving DNA, IgG, IgM rheumatoid factor, and low density lipoproteins. Analysis of the composition of the polyethylene glycol serum precipitates by 2-dimensional gel diffusion, immunoelectrophoresis, and sodium dodecyl sulfate polyacrylamide gel electrophoresis failed to reveal the presence of additional DNA-binding proteins unique to seropositive rheumatoid arthritis. The only feature distinguishing high DNA binding sera from those with low DNA binding activity was an increased amount of polyethylene glycol-insoluble IgG in the former, presumably reflecting IgG/IgG and/or IgG/IgM complexes. The significance of these unusual DNA/low density lipoprotein/IgG/rheumatoid factor complexes with respect to the diagnostic specificity and pathophysiology of the DNA/anti-DNA system is discussed.     

Epithelial junction formation requires confinement of Cdc42 activity by a novel SH3BP1 complex

Epithelial cell-cell adhesion and morphogenesis require dynamic control of actin-driven membrane remodeling. The Rho guanosine triphosphatase (GTPase) Cdc42 regulates sequential molecular processes during cell-cell junction formation; hence, mechanisms must exist that inactivate Cdc42 in a temporally and spatially controlled manner. In this paper, we identify SH3BP1, a GTPase-activating protein for Cdc42 and Rac, as a regulator of junction assembly and epithelial morphogenesis using a functional small interfering ribonucleic acid screen. Depletion of SH3BP1 resulted in loss of spatial control of Cdc42 activity, stalled membrane remodeling, and enhanced growth of filopodia. SH3BP1 formed a complex with JACOP/paracingulin, a junctional adaptor, and CD2AP, a scaffolding protein; both were required for normal Cdc42 signaling and junction formation. The filamentous actin-capping protein CapZ also associated with the SH3BP1 complex and was required for control of actin remodeling. Epithelial junction formation and morphogenesis thus require a dual activity complex, containing SH3BP1 and CapZ, that is recruited to sites of active membrane remodeling to guide Cdc42 signaling and cytoskeletal dynamics.     

Improving the catalytic activity of a thermophilic enzyme at low temperatures

Enzymes from thermophilic organisms often are barely active at low temperatures. To obtain a better understanding of this sluggishness, we used DNA shuffling to mutagenize the trpC gene, which encodes indoleglycerol phosphate synthase, from the hyperthermophile Sulfolobus solfataricus. Mutants producing more active protein variants were selected by genetic complementation of an Escherichia coli mutant bearing a trpC deletion. Single amino acid changes and combinations of these changes improved growth appreciably. Five singly and doubly altered protein variants with changes at the N- and C-termini, or at the phosphate binding site, were purified and characterized with regard to their kinetics of enzymatic catalysis, product binding, cleavage by trypsin, and inactivation by heat. Turnover numbers of the purified variant proteins correlated with the corresponding growth rates, showing that the turnover number was the selected trait. Although the affinities for both the substrate and the product decreased appreciably in most protein variants, these defects were offset by the accumulation of high levels of the enzyme's substrate. Rapid mixing of the product indoleglycerol phosphate with the parental enzyme revealed that the enzyme's turnover number at low temperatures is limited by the dissociation of the enzyme-product complex. In contrast, representative protein variants bind and release the product far more rapidly, shifting the bottleneck to the preceding chemical step. The turnover number of the parental enzyme increases with temperature, suggesting that its structural rigidity is responsible for its poor catalytic activity at low temperatures. In support of this interpretation, the rate of trypsinolysis or of thermal denaturation is accelerated significantly in the activated protein variants.     

Novel alterations in CDK1/cyclin B1 kinase complex formation occur during the acquisition of a polyploid DNA content.

The pathways that regulate the S-phase events associated with the control of DNA replication are poorly understood. The bone marrow megakaryocytes are unique in that they leave the diploid (2C) state to differentiate, synthesizing 4 to 64 times the normal DNA content within a single nucleus, a process known as endomitosis. Human erythroleukemia (HEL) cells model this process, becoming polyploid during phorbol diester-induced megakaryocyte differentiation. The mitotic arrest occurring in these polyploid cells involves novel alterations in the cdk1/cyclin B1 complex: a marked reduction in cdk1 protein levels, and an elevated and sustained expression of cyclin B1. Endomitotic cells thus lack cdk1/cyclin B1-associated H1-histone kinase activity. Constitutive over-expression of cdk1 in endomitotic cells failed to re-initiate normal mitotic events even though cdk1 was present in a 10-fold excess. This was due to an inability of cyclin-B1 to physically associate with cdk1. Nonetheless, endomitotic cyclin B1 possesses immunoprecipitable H1-histone kinase activity, and specifically translocates to the nucleus. We conclude that mitosis is abrogated during endomitosis due to the absence of cdk1 and the failure to form M-phase promoting factor, resulting in a disassociation of mitosis from the completion of S-phase. Further studies on cyclin and its interacting proteins should be informative in understanding endomitosis and cell cycle control.     

The Cik1/Kar3 motor complex is required for the proper kinetochore-microtubule interaction after stressful DNA replication

In budding yeast Saccharomyces cerevisiae, kinetochores are attached by microtubules during most of the cell cycle, but the duplication of centromeric DNA disassembles kinetochores, which results in a brief dissociation of chromosomes from microtubules. Kinetochore assembly is delayed in the presence of hydroxyurea, a DNA synthesis inhibitor, presumably due to the longer time required for centromeric DNA duplication. Some kinetochore mutants are sensitive to stressful DNA replication as these kinetochore proteins become essential for the establishment of the kinetochore-microtubule interaction after treatment with hydroxyurea. To identify more genes required for the efficient kinetochore-microtubule interaction under stressful DNA replication conditions, we carried out a genome-wide screen for yeast mutants sensitive to hydroxyurea. From this screen, cik1 and kar3 mutants were isolated. Kar3 is the minus-end-directed motor protein; Cik1 binds to Kar3 and is required for its motor function. After exposure to hydroxyurea, cik1 and kar3 mutant cells exhibit normal DNA synthesis kinetics, but they display a significant anaphase entry delay. Our results indicate that cik1 cells exhibit a defect in the establishment of chromosome bipolar attachment in the presence of hydroxyurea. Since Kar3 has been shown to drive the poleward chromosome movement along microtubules, our data support the possibility that this chromosome movement promotes chromosome bipolar attachment after stressful DNA replication.     

Dimethyl sulfoxide stimulates the catalytic activity of de novo DNA methyltransferase 3a (Dnmt3a) in vitro

Mammalian DNA methyltransferase Dnmt3a is required for de novo methylation of CpG dinucleotides in genomic DNA. While DNA methyltransferase inhibitors have been extensively utilized both in vitro and in vivo, no stimulator of catalytic activity has been identified thus far. Here we show that the methyltransfer activity of Dnmt3a is stimulated by the addition of dimethyl sulfoxide (DMSO) to the reaction solution in vitro. Enzymatic analysis of initial reaction velocity suggests that the DMSO stimulation effect depends on the interaction between DMSO and the reaction substrates (DNA and AdoMet), but not the enzyme itself.     

Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: Purification and characterization of the inhibitory complex

Saccharomyces cerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the degradation of ornithine decarboxylase (ODC). Despite many studies on the interaction of AZ and ODC, the ODC:AZ complex has not been purified from any source and thus clear information about the stoichiometry of the complex is still lacking. In this study we have studied the yeast antizyme protein and the ODC:AZ complex. The far UV CD spectrum of the full-length antizyme shows that the yeast protein consists of 51% β-sheet, 19% α-helix, and 24% coils. Surface plasmon resonance analyses show that the association constant (K_A) between yeast AZ and yeast ODC is 6 × 10⁷ (M⁻¹). Using purified His-tagged AZ as a binding partner, we have purified the ODC:AZ inhibitory complex. The isolated complex has no ODC activity. The molecular weight of the complex is 90 kDa, which indicates a one to one stoichiometric binding of AZ and ODC in vitro. Comparison of the circular dichroism (CD) spectra of the two individual proteins and of the ODC:AZ complex shows a change in the secondary structure in the complex.     

Crystallization and preliminary X-ray diffraction studies of an a1/α2/DNA ternary complex

Crystals have been obtained of a ternary complex containing the yeast a1/α2 homeodomain heterodimer bound to a 21-base pair DNA site containing two 5′ overhanging bases at each end. The crystals are grown from cobaltic hexamine and form in space group P6₁ or P6₅ with a = b = 133 Å, c = 45.4 Å. Crystals that are flash-frozen at ?179°C diffract to 2.7 Å along the c-axis and to 2.4 Å in perpendicular directions. The crystals contain one protein–DNA complex in the crystallographic asymmetric unit. © 1995 Wiley-Liss, Inc.     

Yeast resolving enzyme CCE1 makes sequential cleavages in DNA junctions within the lifetime of the complex

CCE1 is a DNA junction-resolving enzyme of Saccharomyces cerevisiae. Such enzymes are required to make two symmetrically paired cleavages in order to resolve the four-way junction productively. Using a cruciform assay, we show here that CCE1 introduces two unilateral cleavages in a sequential manner. This requires that the protein remains bound to the junction, preventing branch migration of the point of strand exchange. From a detailed kinetic analysis, we find that the CCE1 cleavage at a given site is accelerated by a factor of 5-10 when it occurs subsequently to the initial cleavage. These properties ensure a productive resolution of the four-way junction and may be general for junction-resolving enzymes.